Singleminded-2 (Sim2) is a member of the bHLH-PAS family of proteins and plays a key role in the developing central nervous system (CNS) by regulating neuron growth. Sim2 was initially identified by positional cloning on chromosome 21 and contributes to many of the physiological abnormalities associated with Downs Syndrome (DS) as a result of trisomy-21. The pattern of occurrence of malignant disorders in people with DS is different with a very high risk of leukemia in childhood extending into young adulthood and a 50% decrease in solid tumors. Women experience a lower risk than men, mainly due to the absence of breast cancer and this is though to be a result of a gene dosage effect of tumor suppressor genes on chromosome 21. Although Sim2 is associated with critical biological functions, very little is known regarding its role in development outside of the CNS, and even less is known about its role in cancer susceptibility. Our hypothesis is that a splice variant of Sim2 (Sim2s) is a mammary tumor suppressor gene required for establishing and/or maintaining normal mammary epithelial cell growth and differentiation. The rationale for this hypothesis is based on preliminary data showing that Sim2s is decreased in primary human breast cancel samples and differentially expressed in human breast epithelial cells and breast cancer cell lines. Re-expression of Sim2s expression in invasive human breast cancer cells decreases proliferation, anchorage-independent growth and invasion. In addition, loss of Sim2s in a virgin mouse mammary gland transplantation model results in abnormal lateral branching, hyperplastic nodules, breakdown of the basement membrane and down-regulation of E-cadherin. The specific aims of this proposal are to: 1) Determine the role of Sim2s in mammary gland development using knockout and transgenic models; 2) The effect of Sim2s loss and overexpression on tumorigenesis using established cancer prone transgenic mice. These results will be significant because they will increase our understanding of a pathway not previously known to be involved in mammary gland development and provide novel targets for preventive, diagnostic and therapeutic treatment of this disease.